Subunit W of photosystem II (PsbW) is a single-span thylakoid membrane protein that is synthesized with a cleavable hydrophobic signal peptide and integrated into the thylakoid membrane by an apparently spontaneous mechanism. In this study, we have analyzed the secondary structure of the pre-protein at early stages of the insertion pathway, using purified recombinant pre-PsbW. We show that the protein remains soluble in Tris buffer after removal of detergent. Under these conditions pre-PsbW contains no detectable α-helix, whereas substantial α-helical structure is present in SDS micelles. In aqueous buffer, the tryptophan fluorescence emission characteristics are intermediate between those of solvent-exposed and hydrophobic environments, suggesting the formation of a partially folded structure. If denaturants are excluded from the purification protocol, pre-PsbW purifies instead as a 180-kDa oligomer with substantial α-helical structure. Mature-size PsbW was prepared by removal of the presequence, and we show that this protein also contains α-helix in detergent but in lower quantities than the pre-protein. We therefore propose that pre-PsbW contains α-helical structure in both the mature protein and the signal peptide in nonpolar environments. We propose that pre-PsbW acquires its α-helical structure only during the later, membrane-bound stages of the insertion pathway, after which it forms a "helical hairpin"-type loop intermediate in the thylakoid membrane.